Heat stable mutants of starch biosynthesis enzymes

ABSTRACT

The subject invention pertains to novel mutant polynucleotide molecules that encode enzymes that have increased heat stability. These polynucleotides, when expressed in plants, result in increased yield in plants grown under conditions of heat stress. The polynucleotide molecules of the subject invention encode maize endosperm ADP glucose pyrophosphorylase (AGP) and soluble starch synthase (SSS) enzyme activties. Plants and plant tissue bred to contain, or transformed with, the mutant polynucleotides, and expressing the polypeptides encoded by the polynucleotides, are also contemplated by the present invention. The subject invention also concerns methods for isolating polynucleotides and polypeptides contemplated within the scope of the invention. Methods for increasing yield in plants grown under conditions of heat stress are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.08/972,545, filed Nov. 18, 1997, now U.S. Pat. No. 6,069,300, whichclaims the benefit of U.S. Provisional Application No. 60/031,045, filedNov. 18, 1996. This application also claims the benefit of U.S.Provisional Application No. 60/085,460, filed May 14, 1998.

This invention was made with government support under National ScienceFoundation grant number 9316887. The government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

The sessile nature of plant life generates a constant exposure toenvironmental factors that exert positive and negative effects on itsgrowth and development. One of the major impediments facing modemagriculture is adverse environmental conditions. One important factorwhich causes significant crop loss is heat stress. Temperature stressgreatly reduces grain yield in many cereal crops such as maize, wheat,and barley. Yield decreases due to heat stress range from 7 to 35% inthe cereals of world-wide importance.

A number of studies have identified likely physiological consequences ofheat stress. Early work by Hunter et al. (Hunter, R. B., Tollenaar, M.,and Breuer, C. M. [1977] Can. J. Plant Sci. 57:1127-1133) using growthchamber conditions showed that temperature decreased the duration ofgrain filling in maize. Similar results in which the duration of grainfilling was adversely altered by increased temperatures were identifiedby Tollenaar and Bruulsema (Tollenaar, M. and Bruulsema, T. W. [1988]Can. J. Plant Sci. 68:935-940). Badu-Apraku et al. (Badu-Apraku, B.,Hunter, R. B., and Tollenaar, M. [1983] Can. J. Plant. Sci. 63:357-363)measured a marked reduction in the yield of maize plants grown under theday/night temperature regime of 35/15° C. compared to growth in a 25/15°C. temperature regime. Reduced yields due to increased temperatures isalso supported by historical as well as climatological studies(Thompson, L. M. [1986] Agron. J. 78:649-653; Thompson, L. M. [1975]Science 188:535-541; Chang, J. [1981] Agricul. Metero. 24:253-262; andConroy, J. P., Seneweera, S., Basra, A. S., Rogers, G., andNissen-Wooller, B. [1994] Aust. J. Plant Physiol. 21:741-758).

That the physiological processes of the developing seed are adverselyaffected by heat stress evident from studies using an in vitro kernelculture system (Jones, R. J., Gengenbach, B. G., and Cardwell, V. B.[1981] Crop Science 21:761-766; Jones, R. J., Ouattar, S., andCrookston, R. K. [1984] Crop Science 24:133-137; and Cheikh, N., andJones, R. J. [1995] Physiol. Plant. 95:59-66). Maize kernels cultured atthe above-optimum temperature of 35° C. exhibited a dramatic reductionin weight.

Work with wheat identified the loss of soluble starch synthase (SSS)activity as a hallmark of the wheat endosperm's response to heat stress(Hawker, J. S. and Jenner, C. F. [1993] Aust. J. Plant Physiol.20:197-209; Denyer, K., Hylton, C. M., and Smith, A. M. [1994] Aust. J.Plant Physiol. 21:783-789; Jenner, C. F. [1994] Aust. J. Plant Physiol.21:791-806). Additional studies with SSS of wheat endosperm show that itis heat labile (Rijven, A. H. G. C. [1986] Plant Physiol. 81:448-453;Keeling, P. L., Bacon, P. J., Holt, D. C. [1993] Planta. 191:342-348;Jenner, C. F., Denyer, K., and Guerin, J. [1995] Aust. J. Plant Physiol.22:703-709).

The roles of SSS and ADP glucose pyrophosphorylase (AGP) under heatstress conditions in maize is less clear. (AGP) catalyzes the conversionof ATP and α-glucose-1-phosphate to ADP-glucose and pyrophosphate.ADP-glucose is used as a glycosyl donor in starch biosynthesis by plantsand in glycogen biosynthesis by bacteria. The importance of ADP-glucosepyrophosphorylase as a key enzyme in the regulation of starchbiosynthesis was noted in the study of starch deficient mutants of maize(Zea mays) endosperm (Tsai, C. Y., and Nelson, Jr., O. E. [1966] Science151:341-343; Dickinson, D. B., J. Preiss [1969] Plant Physiol.44:1058-1062).

Ou-Lee and Setter (Ou-Lee, T. and Setter, T. L. [1985] Plant Physiol.79:852-855) examined the effects of temperature on the apical or tipregions of maize ears. With elevated temperatures, AGP activity waslower in apical kernels when compared to basal kernels during the timeof intense starch deposition. In contrast, in kernels developed atnormal temperatures, AGP activity was similar in apical and basalkernels during this period. However, starch synthase activity duringthis period was not differentially affected in apical and basal kernels.Further, heat-treated apical kernels exhibited an increase in starchsynthase activity over control. This was not observed with AGP activity.Singletary et al. (Singletary, G. W., Banisadr, R., and Keeling, P. L.[1993] Plant Physiol. 102: 6 (suppl).; Singletary, G. W., Banisadra, R.,Keeling, P. L. [1994] Aust. J. Plant Physiol. 21:829-841) using an invitro culture system quantified the effect of various temperaturesduring the grain fill period. Seed weight decreased steadily astemperature increased from 22-36° C. A role for AGP in yield loss isalso supported by work from Duke and Doehlert (Duke, E. R. and Doehlert,D. C. [1996] Environ. Exp. Botany. 36:199-208).

Work by Keeling et al. (1994, supra) quantified SSS activity in maizeand wheat using Q₁₀ analysis, and showed that SSS is an importantcontrol point in the flux of carbon into starch.

In vitro biochemical studies with AGP and SSS clearly show that bothenzymes are heat labile. Maize endosperm AGP loses 96% of its activitywhen heated at 57° C. for five minutes (Hannah, L. C., Tuschall, D. M.,and Mans, R. J. [1980] Genetics 95:961-970). This is in contrast topotato AGP which is fully stable at 70° C. (Sowokinos, J. R. and Preiss,J. [1982] Plant Physiol. 69:1459-1466; Okita, T. W., Nakata, P. A.,Anderson, J. M., Sowokinos, J., Morell, J., and Preiss, J. [1990] PlantPhysiol. 93:785-90). Heat inactivation studies with SSS showed that itis also labile at higher temperatures, and kinetic studies determinedthat the Km value for amylopectin rose exponentially when temperatureincreased from 25-45° C. (Jenner et al., 1995, supra).

Biochemical and genetic evidence has identified AGP as a key enzyme instarch biosynthesis in higher plants and glycogen biosynthesis in E.coli (Preiss, J. and Romeo, T. [1994] Progress in Nuc. Acid Res. and MolBiol. 47:299-329; Preiss, J. and Sivak, M. [1996] “Starch synthesis insinks and sources,” In Photoassimilate distribution in plants and crops:source-sink relationships. Zamski, E., ed., Marcil Dekker Inc. pp.139-168). AGP catalyzes what is viewed as the initial step in the starchbiosynthetic pathway with the product of the reaction being theactivated glucosyl donor, ADPglucose. This is utilized by starchsynthase for extension of the polysaccharide polymer (reviewed inHannah, L. Curtis [1996] “Starch synthesis in the maize endosperm,” In:Advances in Cellular and Molecular Biology of Plants, Vol. 4. B. A.Larkins and I. K. Vasil (eds.). Cellular and Molecular Biology of PlantSeed Development. Kluwer Academic Publishers, Dordrecht, TheNetherlands).

Initial studies with potato AGP showed that expression in E. coliyielded an enzyme with allosteric and kinetic properties very similar tothe native tuber enzyme (Iglesias, A., Barry, G. F., Meyer, C.,Bloksberg, L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W.,Kishore, G. M., and Preiss, J. [1993] J. Biol Chem. 268:1081-86;Ballicora, M. A., Laughlin, M. J., Fu, Y., Okita, T. W., Barry, G. F.,and Preiss, J. [1995] Plant Physiol. 109:245-251). Greene et al.(Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J.,and Okita, T. W. [1996] Proc. Natl. Acad. Sci. 93:1509-1513; Greene, T.W., Woodbury, R. L., and Okita, T. W. [1996] Plant Physiol.(112:1315-1320) showed the usefulness of the bacterial expression systemin their structure-function studies with the potato AGP. Multiplemutations important in mapping allosteric and substrate binding siteswere identified (Okita, T. W., Greene, T. W., Laughlin, M. J., Salamone,P., Woodbury, R., Choi, S., Ito, H., Kavakli, H., and Stephens, K.[1996] “Engineering Plant Starches by the Generation of Modified PlantBiosynthetic Enzymes,” In Engineering Crops for Industrial End Uses,Shewry, P. R., Napier, J. A., and Davis, P., eds., Portland Press Ltd.,London).

AGP enzymes have been isolated from both bacteria and plants. BacterialAGP consists of a homotetramer, while plant AGP from photosynthetic andnon-photosynthetic tissues is a heterotetramer composed of two differentsubunits. The plant enzyme is encoded by two different genes, with onesubunit being larger than the other. This feature has been noted in anumber of plants. The AGP subunits in spinach leaf have molecularweights of 54 kDa and 51 kDa, as estimated by SDS-PAGE. Both subunitsare immunoreactive with antibody raised against purified AGP fromspinach leaves (Copeland, L., J. Preiss (1981) Plant Physiol.68:996-1001; Morell, M., M. Bloon, V. Knowles, J. Preiss [1988] J. Bio.Chem. 263:633). Immunological analysis using antiserum prepared againstthe small and large subunits of spinach leaf showed that potato tuberAGP is also encoded by two genes (Okita et al., 1990, supra). The cDNAclones of the two subunits of potato tuber (50 and 51 kDa) have alsobeen isolated and sequenced (Muller-Rober, B. T., J. Kossmann, L. C.Hannah, L. Willmitzer, U. Sounewald [1990] Mol. Gen. Genet. 224:136-146;Nakata, P. A., T. W. Greene, J. M. Anderson, B. J. Smith-White, T. W.Okita, J. Preiss [1991] Plant Mol. Biol. 17:1089-1093). The largesubunit of potato tuber AGP is heat stable (Nakata et al. [1991],supra).

As Hannah and Nelson (Hannah, L. C., O. E. Nelson (1975) Plant Physiol.55:297-302.; Hannah, L. C., and Nelson, Jr., O. E. [1976] Biochem.Genet. 14:547-560) postulated, both Shrunken-2 (Sh2) (Bhave, M. R., S.Lawrence, C. Barton, L. C. Hannah [1990] Plant Cell 2:581-588) andBrittle-2 (Bt2) (Bae, J. M., M. Giroux, L. C. Hannah [1990] Maydica35:317-322) are structural genes of maize endosperm ADP-glucosepyrophosphorylase. Sh2 and Bt2 encode the large subunit and smallsubunit of the enzyme, respectively. From cDNA sequencing, Sh2 and Bt2proteins have predicted molecular weight of 57,179 Da (Shaw, J. R., L.C. Hannah [1992] Plant Physiol. 98:1214-1216) and 52,224 Da,respectively. The endosperm is the site of most starch deposition duringkernel development in maize. Sh2 and bt2 maize endosperm mutants havegreatly reduced starch levels corresponding to deficient levels of AGPactivity. Mutations of either gene have been shown to reduce AGPactivity by about 95% (Tsai and Nelson, 1966, supra; Dickinson andPreiss, 1969, supra). Furthermore, it has been observed that enzymaticactivities increase with the dosage of functional wild type Sh2 and Bt2alleles, whereas mutant enzymes have altered kinetic properties. AGP isthe rate limiting step in starch biosynthesis in plants. Stark et al.placed a mutant form of E. coli AGP in potato tuber and obtained a 35%increase in starch content (Stark et al. [1992] Science 258:287).

The cloning and characterization of the genes encoding the AGP enzymesubunits have been reported for various plants. These include Sh2 cDNA(Bhave et al., 1990, supra), Sh2 genomic DNA (Shaw and Hannah, 1992,supra), and Bt2 cDNA (Bae et al., 1990, supra) from maize; small subunitcDNA (Anderson, J. M., J. Hnilo, R. Larson, T. W. Okita, M. Morell, J.Preiss [1989] J. Biol. Chem. 264:12238-12242) and genomic DNA (Anderson,J. M., R. Larson, D. Landencia, W. T. Kim, D. Morrow, T. W. Okita, J.Preiss [1991] Gene 97:199-205) from rice; and small and large subunitcDNAs from spinach leaf (Morell et al., 1988, supra) and potato tuber(Muller-Rober et al., 1990, supra; Nakata, P. A., Greene, T. W.,Anderson, J. W., Smith-White, B. J., Okita, T. W., and Preiss, J. [1991]Plant Mol. Biol. 17:1089-1093). In addition, cDNA clones have beenisolated from wheat endosperm and leaf tissue (Olive, M. R., R. J.Ellis, W. W. Schuch [1989] Plant Physiol. Mol. Biol. 12:525-538) andArabidopsis thaliana leaf (Lin, T., Caspar, T., Sommerville, C. R., andPreiss, J. [1988] Plant Physiol. 88:1175-1181).

AGP functions as an allosteric enzyme in all tissues and organismsinvestigated to date. The allosteric properties of AGP were first shownto be important in E. coli. A glycogen-overproducing E. coli mutant wasisolated and the mutation mapped to the structural gene for AGP,designated as glyC. The mutant E. coli, known as glyC-16, was shown tobe more sensitive to the activator, fructose 1,6 bisphosphate, and lesssensitive to the inhibitor, cAMP (Preiss, J. [1984] Ann. Rev. Microbiol.419-458). Although plant AGP's are also allosteric, they respond todifferent effector molecules than bacterial AGP's. In plants,3-phosphoglyceric acid (3-PGA) functions as an activator while phosphate(PO₄) serves as an inhibitor (Dickinson and Preiss, 1969, supra).

Using an in vivo mutagenesis system created by the Ac-mediated excisionof a Ds transposable element fortuitously located close to a knownactivator binding site, Giroux et al. (Giroux, M. J., Shaw, J., Barry,G., Cobb, G. B., Greene, T., Okita, T. W., and Hannah, L. C. [1996]Proc. Natl. Acad. Sci. 93:5824-5829) were able to generate site-specificmutants in a functionally important region of maize endosperm AGP. Onemutant, Rev 6, contained a tyrosine-serine insert and conditioned a11-18% increase in seed weight.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods useful forimproving crop yields in plants, such as those plants that producecereal crops. In one embodiment, the subject invention provides heatstable AGP enzymes and nucleotide sequences which encode these enzymes.In a preferred embodiment, the heat stable enzymes of the invention canbe used to provide plants having greater tolerance to highertemperatures, thus enhancing the crop yields from these plants. In aparticularly preferred embodiment, the improved plant is a cereal.Cereals to which this invention applies include, for example, maize,wheat, rice, and barley.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows heat stable maize endosperm AGP large subunit mutants.Percentage of AGP activity remaining after five minutes of heattreatment at 60° C is shown.

FIG. 2 shows primary sequence alignment of the region surrounding HS 33mutation in the AGP large subunits for maize, wheat, barley, and potato.Conserved regions are boxed.

FIG. 3 shows primary sequence alignment of the region surrounding HS 40mutation in the AGP large subunits for maize, wheat, barley, and potato.Conserved regions are boxed. Bolded aspartic acid residue corresponds toD413A allosteric mutant of potato LS (Greene, T. W., Woodbury, R. L.,and Okita, T. W. [1996] Plant Physiol. (112:1315-1320). Spinach leaf AGPsequence is the activator site 2 peptide identified in 3-PGA analoguestudies (Ball, K. and Preiss, J. [1994] J. Biol. Chem. 269:24706-24711).The labeled lysine residue is bolded.

FIGS. 4A and 4B show molecular characterization of TS48 and TS60,respectively. Genetic lesion of TS48 and corresponding residues are inbold. The amino acid number is indicated above the Leu to Phe mutationof TS48. The last line is a consensus sequence. The Leu residue ishighly conserved. Genetic lesions of TS60 and corresponding residues arein bold. The amino acid numbers are indicated above the Glu to Lys andAla to Val mutations of TS60. Boxed residues correspond to the HS 33mutation previously identified and shown to be important in heatstability of the maize endosperm AGP. The last line is a consensussequence.

FIGS. 5A and 5B show molecular characterization of RTS 48-2 and RTS60-1, respectively. Genetic lesion of RTS 48-2 and correspondingresidues are in bold. The amino acid number is indicated above the Alato Val mutation of RTS 48-2. The last line is a consensus sequence. Ofsignificance, the mutation identified in RTS 48-2 maps to the identicalresidue found in the heat stable variant HS13. HS 13 contained an Ala toPro mutation at position 177. Genetic lesion of RTS 60-1 andcorresponding residues are in bold. The amino acid number is indicatedabove the Ala to Val mutation of RTS 60-1. The last line is a consensussequence.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is an amino acid sequence of a region of the large subunitof AGP in maize containing the HS33 mutation as shown in FIG. 2.

SEQ ID NO. 2 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 2.

SEQ ID NO. 3 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 2.

SEQ ID NO. 4 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 2.

SEQ ID NO. 5 is an amino acid sequence of a region of the large subunitof AGP in potato as shown in FIG. 2.

SEQ ID NO. 6 is an amino acid sequence of a region of the large subunitof AGP in maize containing the HS40 mutation in FIG. 3.

SEQ ID NO. 7 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 3.

SEQ ID NO. 8 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 3.

SEQ ID NO. 9 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 3.

SEQ ID NO. 10 is an amino acid sequence of a region of the large subunitof AGP in potato as shown in FIG. 3.

SEQ ID NO. 11 is an amino acid sequence of a region of the large subunitof AGP in spinach as shown in FIG. 3.

SEQ ID NO. 12 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS48 mutation as shown in FIG. 4A.

SEQ ID NO. 13 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4A.

SEQ ID NO. 14 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4A.

SEQ ID NO. 15 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4A.

SEQ ID NO. 16 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4A.

SEQ ID NO. 17 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS60 mutation as shown in FIG. 4B.

SEQ ID NO. 18 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4B.

SEQ ID NO. 19 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4B.

SEQ ID NO. 20 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4B.

SEQ ID NO. 21 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4B.

SEQ ID NO. 22 is an amino acid sequence of a region of the large subunitof AGP in maize containing the TS60 as shown in FIG. 4B.

SEQ ID NO. 23 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 4B.

SEQ ID NO. 24 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 4B.

SEQ ID NO. 25 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 4B.

SEQ ID NO. 26 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 4B.

SEQ ID NO. 27 is an amino acid sequence of a region of the large subunitof AGP in maize containing the RTS48-2 as shown in FIG. 5A.

SEQ ID NO. 28 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 5A.

SEQ ID NO. 29 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 5A.

SEQ ID NO. 30 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 5A.

SEQ ID NO. 31 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 5A.

SEQ ID NO. 32 is an amino acid sequence of a region of the large subunitof AGP in maize containing the RTS60-1 as shown in FIG. 5B.

SEQ ID NO. 33 is an amino acid sequence of a region of the large subunitof AGP in maize as shown in FIG. 5B.

SEQ ID NO. 34 is an amino acid sequence of a region of the large subunitof AGP in wheat as shown in FIG. 5B.

SEQ ID NO. 35 is an amino acid sequence of a region of the large subunitof AGP in barley as shown in FIG. 5B.

SEQ ID NO. 36 is an amino acid sequence of a region of the large subunitof AGP in rice as shown in FIG. 5B.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns novel mutant polynucleotide molecules,and the polypeptides encoded thereby, that confer increased yield inplants grown under conditions of heat stress relative to plants havingwild type genotype. In specific embodiments, the polynucleotidemolecules of the subject invention encode maize endosperm ADP glucosepyrophosphorylase (AGP) and soluble starch synthase (SSS) enzymeactivities. The mutant enzymes confer increased stability to heat stressconditions during seed and plant development in seeds and plant tissueexpressing the enzymes as compared with wild type enzyme activities.

In one embodiment, a polynucleotide of the present invention encodes amutant large subunit of AGP containing a histidine-to-tyrosine aminoacid substitution in the sequence of the polypeptide. This substitutionoccurs at amino acid residue number 333, according to the acceptednumber of the amino acids in this protein (Shaw and Hannah, 1992,supra). The position of this substitution can be readily identified by aperson skilled in the art. A second mutation exemplified in the subjectinvention is a threonine-to-isoleucine substitution at position number460 of the AGP protein. Additional exemplified mutants conferringincreased heat stability are shown below in Table 1.

TABLE 1 Mutant Amino Acid Change HS 13 Ala to Pro at position 177 HS 14Asp to His at position 400, and Val to Ile at position 454 HS 16 Arg toThr at position 104 HS 33 His to Tyr at position 333 HS 39 His to Tyr atposition 333 HS 40 His to Tyr at position 333, and Thr to Ile atposition 460 HS 47 Arg to Pro at position 216, and His to Tyr atposition 333 RTS 48-2 Ala to Val at position 177 RTS 60-1 Ala to Val atposition 396

cDNA clones for the subunits of the maize endosperm AGP (SH2 and BT2)and an E. coli strain deficient in the endogenous bacterial AGP (glg C⁻)(AC70R1-504) have facilitated the establishment of a bacterialexpression system to study the maize endosperm AGP. Expression of asingle subunit is unable to complement the glg C⁻ mutant, and noglycogen is produced (Iglesias, A., Barry, G. F., Meyer, C., Bloksberg,L., Nakata, P., Greene, T., Laughlin, M. J., Okita, T. W., Kishore, G.M., and Preiss, J. [1993] J. Biol Chem. 268: 1081-86). However,expression of both the large and small subunits on compatible expressionvectors fully complements the glg C⁻ mutation and restores glycogenproduction as evidenced by a dark, reddish-brown staining of coloniesexposed to iodine. Thus, complementation is easily identified by simplyexposing the colonies to iodine.

In one embodiment, E. coli glg C⁻ cells expressing the structural genesfor either potato or maize endosperm AGP were used. Cells containingpotato AGP genes can synthesize copious levels of glycogen when grown at37° or at 42° C. However, cells expressing maize endosperm AGP onlysynthesize glycogen at 37° C. This result demonstrates the heatsensitivity of wild-type maize endosperm AGP. That there is a differencebetween potato and maize AGP's in this regard provides an efficientsystem for screening for mutant cells that have heat stable variants ofthe maize endosperm AGP.

One aspect of the subject invention pertains to the efficientidentification of AGP which is heat stable. Accordingly, a plasmidcomprising a polynucleotide coding for the SH2 subunit of maize AGP waschemically mutagenized, as described below, placed into mutant E. colicells expressing the BT2 subunit, and the cells grown at 42° C. toselect for mutants that could produce glycogen at that temperature.Other mutagens known in the art can also be used. Eleven heritable,iodine staining mutants, termed heat stable (HS) mutants, were isolated.Crude extracts of these mutants were prepared and the heat stability ofthe resulting AGP was monitored. The mutants retained between 8-59% oftheir activity after incubation at 60° C. for five minutes (FIG. 1).This compares to the 1-4% routinely observed for wild-type AGP at thistemperature.

The results show that heat stable forms of enzymes can be createdaccording to the subject invention by mutation. Thus, one aspect of theinvention pertains to processes for producing and identifyingpolynucleotides encoding mutant starch biosynthesis enzymes havingincreased heat stability compared to wild type enzymes. Unexpectedly,total activity of the maize endosperm AGP before heat treatment waselevated about two- to three-fold in the majority of these mutants. Thissurprising result makes these mutants particularly advantageous for usein agriculture. Mutagenesis techniques as described herein can be usedaccording to the subject invention to identify other genes encoding heatstable starch biosynthesis enzymes.

The genes encoding several of the heat stable mutants exemplifiedherein, including the two most heat stable HS mutants, HS 33 and HS 40,were completely sequenced. HS 33, which retains 59% of its activityafter heat treatment, contains a single base pair mutation that changesa histidine residue at position 333 of the amino acid sequence of thepolypeptide to a tyrosine (FIG. 2). Primary sequence alignments with thelarge subunits from wheat and barley AGPs show that a histidine is alsopresent at the analogous residue (FIG. 3) (Ainsworth, C., Hosein, F.,Tarvis, M., Weir, F., Burrell, M., Devos, K. M., Gale, M. D. [1995]Planta 197:1-10). Sequence analysis of HS 40, which retains 41% of itsactivity post heat treatment, also contained a histidine to tyrosinemutation at position 333. An additional point mutation was identifiedthat generated a threonine to isoleucine substitution. The threonineresidue is highly conserved in AGP large subunits, while in AGP smallsubunits the analogous residue is either a cysteine or serine (Ainsworthet al., 1995, supra). The threonine to isoleucine substitution islocated close to the carboxyl terminus of the large subunit, and closeto a known binding site for the activator 3-PGA (FIG. 3).

Another aspect of the present invention pertains to mutant starchbiosynthesis enzymes, such as AGP, and the polynucleotides that encodethem, wherein these mutant enzymes are isolated by selecting fortemperature sensitive (TS) mutants which are then mutagenized andscreened for revertants that show enhanced stability. A further aspectof the invention concerns the methods for producing and identifying thepolynucleotides and mutant enzymes encoded thereby.

The subject invention also concerns heat stable mutants of AGP that havemutations in the small subunit of the enzyme. Also encompassed withinthe scope of the invention are polynucleotides that encode the mutantsmall subunits of AGP. Mutations in the small subunit of AGP that conferheat stability to the enzyme can also be readily prepared and identifiedusing the methods of the subject invention.

Plants and plant tissue bred to contain or transformed with the mutantpolynucleotides, and expressing the polypeptides encoded by thepolynucleotides, are also contemplated by the present invention. Plantsand plant tissue expressing the mutant polynucleotides produce tissuesthat have, for example, lower heat-induced loss in weight or yield whensubjected to heat stress during development.

The subject invention also concerns methods for producing andidentifying polynucleotides and polypeptides contemplated within thescope of the invention. In one embodiment, gene mutation, followed byselection using a bacterial expression system, can be used to isolatepolynucleotide molecules that encode enzymes that can alleviateheat-induced loss in starch synthesis in plants.

The subject invention further concerns plants and plant tissue that havean AGP mutant gene incorporated into its genome. Other alleles disclosedherein can also be incorporated into a plant genome. In a preferredembodiment, the plant is a cereal plant. More preferably, the plant isZea mays. Plants having an AGP mutant gene can be grown from seeds thatcomprise a mutant gene in their genome. In addition, techniques fortransforming plants with a gene are known in the art.

Because of the degeneracy of the genetic code, a variety of differentpolynucleotide sequences can encode each of the variant AGP polypeptidesdisclosed herein. In addition, it is well within the skill of a persontrained in the art to create alternative polynucleotide sequencesencoding the same, or essentially the same, polypeptides of the subjectinvention. These variant or alternative polynucleotide sequences arewithin the scope of the subject invention. As used herein, references to“essentially the same” sequence refers to sequences which encode aminoacid substitutions, deletions, additions, or insertions which do notmaterially alter the functional activity of the polypeptide encoded bythe AGP mutant polypeptide described herein.

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, wouldencompass known analogs of natural nucleotides that can function in asimilar manner as naturally-occurring nucleotides. The polynucleotidesequences include both the DNA strand sequence that is transcribed intoRNA and the RNA sequence that is translated into protein. Thepolynucleotide sequences include both full-length sequences as well asshorter sequences derived from the full-length sequences. It isunderstood that a particular polynucleotide sequence includes thedegenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell. Allelicvariations ofthe exemplified sequences also come within the scope of thesubject invention. The polynucleotide sequences falling within the scopeof the subject invention further include sequences which specificallyhybridize with the exemplified sequences. The polynucleotide includesboth the sense and antisense strands as either individual strands or inthe duplex.

Substitution of amino acids other than those specifically exemplified inthe mutants disclosed herein are also contemplated within the scope ofthe present invention. Amino acids can be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a mutant AGP polypeptide having an amino acid ofone class is replaced with another amino acid of the same class fallwithin the scope of the subject invention so long as the mutant AGPpolypeptide having the substitution still retains increased heatstability relative to a wild type polypeptide. Table 2 below provides alisting of examples of amino acids belonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

For example, substitution of the tyrosine at position 333 in the HS 33,HS 39, HS 40 and HS 47 mutant maize endosperm AGP with other aminoacids, such as Glycine, Serine, Threonine, Cysteine, Asparagine, andGlutamine, are encompassed within the scope of the invention.

The subject invention also concerns polynucleotides which encodefragments of the full length mutant polypeptide, so long as thosefragments retain substantially the same functional activity as fulllength polypeptide. The fragments of mutant AGP polypeptide encoded bythese polynucleotides are also within the scope of the presentinvention.

The subject invention also contemplates those polynucleotide moleculesencoding starch biosynthesis enzymes having sequences which aresufficiently homologous with the wild type sequence so as to permithybridization with that sequence under standard high-stringencyconditions. Such hybridization conditions are conventional in the art(see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1989] MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

The polynucleotide molecules of the subject invention can be used totransform plants to express the mutant heat stable enzyme in thoseplants. In addition, the polynucleotides of the subject invention can beused to express the recombinant variant enzyme. They can also be used asa probe to detect related enzymes. The polynucleotides can also be usedas DNA sizing standards.

The polynucleotide molecules of the subject invention also include thosepolynucleotides that encode starch biosynthesis enzymes, such as AGPenzymes, that contain mutations that can confer increased seed weight,in addition to enhanced heat stability, to a plant expressing thesemutants. The combination of a heat stabilizing mutation, such as forexample HS 33 or HS 40, with a mutation conferring increased seedweight, e.g., Rev 6, is specifically contemplated for the presentinvention. See, for example, U.S. Pat. Nos. 5,589,618 and 5,650,557regarding mutations that confer increased seed weight.

Mutations in the AGP subunits that confer heat stability can be combinedaccording to the subject invention with phosphate insensitive mutants ofmaize, such as the Rev6 mutation, to enhance the stability of the Rev6encoded large subunit.

It is expected that enzymic activity of SSS will be impaired at highertemperatures as observed with AGP. Thus, mutagenized forms of the can beexpressed under increased thermal conditions (42° C.), to isolate heatstable variants in accordance with the methods described herein. Theseheat stable mutagenized forms of SSS are further aspects of the subjectinvention.

All public ations and patents cited herein are hereby incorporated byreference.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1

Use of Mutagenesis to Obtain Maize Endosoerm AGP Heat Stable Variants

The chemical mutagen hydroxylamine-HCl was initially used for the randommutagenesis of the large subunit expression plasmid. Hydroxylaminepreferentially hydroxylates the amino nitrogen at the C-4 position ofcytosine, and leads to a GC to AT transition (Suzuki, D. T., Griffith,A. J. F., Miller, J. H., and Lewontin, R. C. [1989] In Introduction togenetic analysis, Freeman, N.Y., 4th ed., pp. 475-499). The chemicalmutagen was chosen for its high mutation frequency. Limitations of thechemical mutagen are recognized, and if a large variety of geneticvariants are not isolated, PCR based random mutagenesis can beperformed. PCR mutagenesis generates a broader spectrum of mutationsthat include similar frequencies of transitions and transversion, andprovides an excellent alternative to the chemical method. The methodoutlined by Cadwell and Joyce (Cadwell, R. C. and Joyce, G. F. [1992]PCR Methods and Applications 2:28-33) can be followed for the PCR basedmethod.

Since the complete expression plasmid is used in the random mutagenesis,it is possible that mutations will occur outside of the coding region.Although it is expected that such mutations will not have any effect onthe heat stability of the maize endosperm AGP, each variant can besubcloned into an unmutated expression plasmid before any additionalcharacterization at the enzyme level is conducted. Both the large andsmall subunit expression plasmids can be constructed so that a NcoI/SacIdigestion will liberate the complete coding region. This can easily becloned back into a unmutated NcoI/SacI digested expression plasmid.

EXAMPLE 2

Molecular Characterization and Analysis of Heat Stable AGP Variants

Initially, 11 heat stable variants of the maize endosperm large subunitwere obtained. Sequencing was performed using DuPont and ABIinstrumentation. Sequence data can be routinely compared to theprogenitor wild-type allele. This analysis reveals the extent ofdiversity of changes conditioning heat stability.

Several of the sequenced HS mutants contained the identical histidine totyrosine change at amino acid position 333 in the large subunit.PCR-derived HS mutants can be quickly screened for the histidine totyrosine alteration by use of site-specific mutagenesis using primersthat change the tyrosine back to histidine.

EXAMPLE 3

Expression, Purification and Kinetic Analysis of Genetic Variants

Conditions for the expression of the wild-type maize endosperm AGP in E.coli have been fully characterized. Optimum growth and inductionconditions vary somewhat from those previously published for potato AGPexpressed in E. coli (Iglesias et al., 1993, supra; Ballicora et al.,1995, supra). Induction at room temperature for 12-14 hrs in thepresence of 0.3 mM IPTG and 25 μg/ml nalidixic acid consistently giveshigh levels of expression and activity. Addition of 30% ammonium sulfateand 10 mM KH₂PO₄ ⁻/K₂HPO₄ ⁻ to the extraction buffer stabilizes themaize AGP in the crude extract.

Ammonium sulfate concentrated AGP is further purified by HydrophobicInteraction Chromatography using Tentacle C3 aminopropyl media (EMSeparations) packed into a Pharmacia HR 10/10 column. Protein binds tothe column in a buffer containing 1 M ammonium sulfate. AGP is elutedfrom the column by successive step gradient washes of buffer thatcontains 0.75 M, 0.5 M, 0.25 M, and 0 M ammonium sulfate. Wild-typemaize endosperm AGP typically elutes in the 0.25 M wash. C3 purifiedmaize endosperm AGP is further purified by anion exchange chromatographyusing Macro-Prep DEAE (BioRad) anion exchange media packed into aPharmacia HR 10/10 column. AGP is eluted by a linear gradient of 100-500mM KCl, and typically elutes at a salt concentration around 300 mM. APharmacia FPLC system is used for all chromatography steps. Theconditions for the individual purification steps are fullycharacterized. AGP activity during the purification is monitored by thepyrophosphorylysis assay, and purification steps are monitored bySDS-PAGE, Coomassie staining, and Western analysis using polyclonalantibodies specific to the maize endosperm AGP large and small subunits.

EXAMPLE 4

Enhanced Subunit Interaction

A totally unexpected pleiotropic effect of the HS maize endosperm AGPmutants is a two- to three-fold elevation of activity before heattreatment. One possible explanation for this result is that we have, bymutational change, shifted the ratio of SH2 and BT2 monomers andpolymers existing within the E. coli cell. Perhaps, in wild-type, only10% or less of the total proteins exist in the active heterotetramericform whereas in the mutants, this percentage is much higher. If thepolymer is more heat resistant than are the monomers, then the phenotypeof the mutants would be identical to what has been observed. Kineticanalysis can be used to determine changes in affinities for substratesand/or allosteric effectors.

To test the idea that the monomer/polymer ratio may be altered in thesemutants, the amounts of monomers and polymers in wild-type and inselected mutants both before and after heat treatment can be monitored.The availability of antibodies (Giroux, M. J., and Hannah, L. C. [1994]Mol. Gen. Genetics 243:400-408) for both subunits makes this approachfeasible. This can be examined both through sucrose gradientultracentrifugation and through gel chromatography and will readilydetermine which method is most efficient and definitive.

Since the higher plant AGP consists of two similar but distinct subunitsthat oligomerize to form the native heterotetrameric structure,mutations that enhance this interaction can provide added stability tothe enzyme. A yeast two-hybrid system (CLONTECH Laboratories, Palo Alto,Calif.) can be used to evaluate subunit interactions. Specific primersfor the amplification of the coding regions can be constructed. Theseprimers add unique restriction sites to the 5′- and 3′-ends so thatcloning facilitates the translational fusion of the individual subunitto the GAL4 DNA binding domain (pGBT9) or GAL4 activation domain(pGAD424). If the proteins cloned into the vectors interact, the DNAbinding domain and the activation domain will form a functionaltranscription activator. This in turn activates expression of thereporter gene, lac Z, cloned behind a GAL4 promoter.

Initially, conditions can be characterized with the wild-type subunits.The coding regions of the wild-type large and small subunits can becloned into the pGBT9 and pGAD424 yeast expression vectors. All possiblecombinations can be generated and tested. pGBT9 and pGAD424 vectorscontaining Sh2 and Bt2 can be cotransformed into the same yeast strain,and selected for growth on media lacking tryptophan (pGBT9) and leucine(pGAD424). Subunit interaction as a function of lacZ expression can bedetected two ways. Positive colonies are visually identified by aB-galactosidase filter assay. With this assay colonies are bound to thefilter, lysed, and incubated with an X-gal solution. Colonies thatexhibit a blue color can be analyzed. Subunit interaction can be furtheranalyzed by an enzyme assay specific for B-galactosidase. This allowsthe quantification of the interaction. Mutations that enhance subunitinteractions will give higher levels of B-galactosidase activity whenassayed.

EXAMPLE 5

Further Enhancement of Stability

The large subunit mutants isolated vary in their heat stabilitycharacteristics, suggesting the possibility of multiple mutations. Whilesequence analysis of mutants HS 33 and HS 40 reveal that the mutantsequences are not identical, both mutants contained the identicalhistidine to tyrosine change. Given the identification of different HSalterations within the SH2 protein, it is possible to efficientlypyramid these changes into one protein. Furthermore, any HS mutationswithin the small subunit can be co-expressed with HS SH2 mutants tofurther enhance the stability of the maize endosperm enzyme.

Multiple HS mutants within one subunit can easily be combined. Forexample, different unique restriction sites that divide the codingregions of Sh2 into three distinct fragments can be used. Whereappropriate, mutation combinations can be generated by subcloning thecorresponding fragment containing the added mutation. If two mutationsare in close proximity, then site-directed mutagenesis can be used toengineer such combinations. One method for site specific mutationsinvolves PCR, mutagenic primer, and the use of DpnI restrictionendonuclease. Primers can be constructed to contain the mutation in the5′ end, and used to PCR amplify using the proofreading polymerase Vent.Amplified DNA can then be digested with DpnI. Parental DNA isolated fromE. coli is methylated and hence susceptible to DpnI. Digested DNA issize fractionated by gel electrophoresis, ligated, and cloned into theexpression vectors. Mutations are confirmed by sequence analysis andtransformed into the AC70R1-504 strain carrying the wild-type smallsubunit. Combinatorial mutants can then be analyzed.

EXAMPLE 6

Combination of Heat Stability Mutations with Rev6

According to the subject invention, the heat stable mutations can becombined with a mutation associated with increased seed weight, such as,for example, the Rev6 mutation. The goal is to maintain the desiredphosphate insensitivity characteristic of Rev6 while enhancing itsstability. Rev 6/HS double mutants can be constructed and confirmed asdescribed herein. Double mutants can be transformed into AC70R1-504carrying the wild-type small subunit. Increased heat stability can beeasily identified by a positive glycogen staining on a low glucosemedia. Rev6 does not stain when grown on this media. Initially allmutant combinations can be screened enzymatically for maintenance ofphosphate insensitivity, and only combinations that maintain phosphateinsensitivity are further analyzed.

EXAMPLE 7

Cloning of SSS I Mutants

A glg A⁻ E. coli strain deficient in the endogenous bacterial glycogensynthase can be obtained from the E. coli Stock Center. Bacterialexpression vectors currently used for the expression of AGP can be usedfor expression of SSS.

One cloning strategy, as used, for example, with Sh2 and Bt2 (Giroux etal., 1996, supra), is the following: One primer contains a uniquerestriction plus the 5′ terminus of the transcript while the otherprimer contains another unique restriction site and sequences 3′ to thetranslational termination codon of the gene under investigation.Subsequent cloning of these gives rise to a translational fusion withinthe plasmid. These gene specific primers are initially used in RT-PCRreactions using poly A+RNA from developing endosperms.

Expression of the maize endosperm SSS I will complement the lack ofglycogen synthase activity in the glg A⁻ strain. Complementation shouldbe easily visualized with iodine staining as it is with the expressionof AGP in the glg C⁻ strain. Crude extracts can be incubated at varioustemperatures and lengths of time to determine the heat stability of SSSI. The glg A⁻ strain expressing the maize endosperm SSS I can be grownat various temperatures to determine if finction is temperaturesensitive as it is with the AGP bacterial expression system. Once arestrictive temperature is established, a random mutagenesis can beconducted with the SSS I clone. Mutant forms of SSS I can be transformedinto the glg A⁻ strain, grown at the restrictive temperature, and heatstable variants identified by their ability to produce iodine-stainingglycogen at the restriction temperature.

EXAMPLE 8

Temperature Sensitive Mutants of Maize Endosperm ADP-glucosePyrophosphorylase

As an alternative approach to identify additional variants withincreased stability, a reverse-genetics approach was employed.Temperature sensitive (TS) mutants have been isolated. These mutantsexhibit a negative iodine staining phenotype at 30° C. indicating a lackof function with the maize endosperm AGP. In contrast, when the mutantsare grown at 37° C. they can fully complement the mutation in thebacterial AGP. This clearly shows that the mutant AGPs are functional,and that the loss of function is temperature dependent. Wild type AGPexhibits a positive glycogen staining phenotype at 30 and 37° C. Thetemperature sensitive mutants were then used to produce second siterevertants that encode mutant AGP having enhanced stability.

Mutagenesis. pSh2 DNA was subjected to hydroxylamine mutagenesis(Greene, T. W., Chantler, S. E., Kahn, M. L., Barry, G. F., Preiss, J.,and Okita, T. W. [1996] Proc. Natl. Acad. Sci. 93:1509-1513) andtransformed into AC70R1-504 E. coli cells carrying the wild type pBt2small subunit plasmid. Cells were plated and grown at 30° C. Temperaturesensitive variants of AGP were identified by their negative iodinestaining phenotype at 30° C. Putative mutants were streaked again at 30°C. and 37° C. along with the wild type AGP as a control. Six mutantsthat consistently gave a negative iodine phenotype at 30° C. and apositive iodine staining phenotype at 37° C. were isolated. Expressionof wild type Sh2 and Bt2 gave a positive iodine staining phenotype atboth temperatures.

Characterization of TS48 and TS60. Plasmid DNA from two temperaturesensitive mutants, TS48 and TS60, was isolated and sequenced to identifythe genetic lesion. A single point mutation that generated thereplacement of leucine at amino acid position 426 with phenylalanine wasidentified (FIG. 4A). This residue and surrounding region is highlyconserved in the cereal endosperm large subunits (LS) (Smith-White andPreiss, 1992). In TS60, two point mutations were identified thatgenerated a glutamic acid to lysine change at amino acid 324 and analanine to valine mutation at position 359 (FIG. 4B). Glu-324 is highlyconserved among the LS and small (SS) subunits of AGP (Smith-White andPreiss, 1992). Ala-359 and the surrounding amino acids are also highlyconserved among the AGP LS. Of significance, the two mutation identifiedin TS60 flank the HS 33 mutation described herein. The HS 33 mutation,which has the histidine to tyrosine substitution at position 333, wasshown to greatly enhance heat stability of the maize endosperm AGP. Thatthe mutations of TS60 are in close proximity to the HS 33 mutation isadditional evidence that this region of the protein is important forstability.

Isolation of second-site revertants. Isolation of the temperaturesensitive mutants provides a selectable phenotype for isolatingadditional variants that enhance the stability of AGP. Additionalhydroxylamine mutagenesis was conducted with TS48 and TS60 DNA toisolate second-site revertants that restore a positive glycogen stainingphenotype at 30° C. Hydroxylamine was used because the chemistry of themutagenesis eliminates the possibility of a direct reversion of theprimary mutation identified in the TS48 and TS60 mutants. This forcesthe selection of second-site mutations that can restore stability inthese temperature sensitive mutants.

Three revertants were isolated for TS48 and the molecularcharacterization of one mutant, RTS 48-2, is shown (FIG. 5A). RTS 48-2contains an alanine to valine mutation at amino acid position 177 inaddition to the parental mutation identified in TS48. This residue andthe surrounding region are highly conserved. The RTS 48-2 mutationcorresponds to the identical site of the mutation identified in the heatstable mutant, HS 13. The alanine residue was mutated to a proline atposition 177 in HS 13. That these two mutations map to the same site issignificant. The RTS 48-2 and HS 13 mutants were selected based onincreased stability using completely different approaches, and thusthese two mutations identify this site to be important in the stabilityof AGP.

Five second-site revertants were isolated for TS60 and the sequenceanalysis of one, RTS 60-1, is shown (FIG. 5B). An alanine to valinemutation at amino acid 396 was identified. This residue is highlyconserved among the AGP LS, and it also maps close to a heat stablemutation identified in HS 14.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

36 1 33 PRT HS33 Mutant of Zea mays 1 Leu His Asp Phe Gly Ser Glu IleLeu Pro Arg Ala Val Leu Asp Tyr 1 5 10 15 Ser Val Gln Ala Cys Ile PheThr Gly Tyr Trp Glu Asp Val Gly Thr 20 25 30 Ile 2 33 PRT Zea mays 2 LeuHis Asp Phe Gly Ser Glu Ile Leu Pro Arg Ala Val Leu Asp His 1 5 10 15Ser Val Gln Ala Cys Ile Phe Thr Gly Tyr Trp Glu Asp Val Gly Thr 20 25 30Ile 3 33 PRT Triticum aestivum 3 Leu His Asp Phe Gly Ser Glu Ile Leu ProArg Ala Leu His Asp His 1 5 10 15 Asn Val Gln Ala Tyr Val Phe Thr AspTyr Trp Glu Asp Ile Gly Thr 20 25 30 Ile 4 33 PRT Hordeum vulgare 4 LeuHis Asp Phe Gly Ser Glu Ile Leu Pro Arg Ala Leu His Asp His 1 5 10 15Asn Val Gln Ala Tyr Val Phe Thr Asp Tyr Trp Glu Asp Ile Gly Thr 20 25 30Ile 5 33 PRT Solanum tuberosum 5 Ser Asn Asp Phe Gly Ser Glu Ile Leu ProAla Ala Ile Asp Asp Tyr 1 5 10 15 Asn Val Gln Ala Tyr Ile Phe Lys AspTyr Trp Glu Asp Ile Gly Thr 20 25 30 Ile 6 26 PRT HS40 Mutant of Zeamays 6 Ala Gly Lys Val Pro Ile Gly Ile Gly Arg Asn Ile Lys Ile Arg Asn 15 10 15 Cys Ile Ile Asp Met Asn Ala Arg Ile Gly 20 25 7 26 PRT Zea mays7 Ala Gly Lys Val Pro Ile Gly Ile Gly Arg Asn Thr Lys Ile Arg Asn 1 5 1015 Cys Ile Ile Asp Met Asn Ala Arg Ile Gly 20 25 8 26 PRT Triticumaestivum 8 Glu Gly Lys Val Pro Ile Gly Val Gly Glu Asn Thr Lys Ile SerAsn 1 5 10 15 Cys Ile Ile Asp Met Asn Ala Arg Ile Gly 20 25 9 26 PRTHordeum vulgare 9 Glu Gly Lys Val Pro Ile Gly Val Gly Glu Asn Thr LysIle Ser Asn 1 5 10 15 Cys Ile Ile Asp Met Asn Ala Arg Ile Gly 20 25 1026 PRT Solanum tuberosum 10 Glu Gly Lys Val Pro Ile Gly Ile Gly Glu AsnThr Lys Ile Arg Lys 1 5 10 15 Cys Ile Ile Asp Lys Asn Ala Lys Ile Gly 2025 11 11 PRT Spinicia oleracea 11 Ile Lys Asp Ala Ile Ile Asp Lys AsnAla Arg 1 5 10 12 20 PRT TS48 Mutant of Zea mays 12 Cys Ser Arg Val SerSer Gly Cys Glu Phe Lys Asp Ser Val Met Met 1 5 10 15 Gly Ala Asp Ile 2013 20 PRT Zea mays 13 Cys Ser Arg Val Ser Ser Gly Cys Glu Leu Lys AspSer Val Met Met 1 5 10 15 Gly Ala Asp Ile 20 14 20 PRT Triticum aestivum14 Arg Ser Arg Leu Asn Ser Gly Ser Glu Leu Lys Asn Ala Met Met Met 1 510 15 Gly Ala Asp Ser 20 15 20 PRT Hordeum vulgare 15 Arg Ser Arg LeuAsn Ser Gly Ser Glu Leu Lys Asn Ala Met Met Met 1 5 10 15 Gly Ala AspSer 20 16 12 PRT Oryza sativa 16 Ser Ser Arg Val Ser Ser Gly Cys Glu LeuLys Ile 1 5 10 17 24 PRT TS60 Mutant of Zea mays 17 Asp Phe Gly Ser LysIle Leu Pro Arg Ala Val Leu Asp His Ser Val 1 5 10 15 Gln Ala Cys IlePhe Thr Gly Tyr 20 18 24 PRT Zea mays 18 Asp Phe Gly Ser Glu Ile Leu ProArg Ala Val Leu Asp His Ser Val 1 5 10 15 Gln Ala Cys Ile Phe Thr GlyTyr 20 19 24 PRT Triticum aestivum 19 Asp Phe Gly Ser Glu Ile Leu ProArg Ala Leu His Asp His Asn Val 1 5 10 15 Gln Ala Tyr Val Phe Thr AspTyr 20 20 24 PRT Hordeum vulgare 20 Asp Phe Gly Ser Glu Ile Leu Pro ArgAla Leu His Asp His Asn Val 1 5 10 15 Gln Ala Tyr Val Phe Thr Asp Tyr 2021 25 PRT Oryza sativa 21 Asp Phe Gly Ser Glu Ile Leu Pro Arg Ala LeuLeu Glu His Asn Val 1 5 10 15 Lys Val Ala Cys Val Phe Thr Glu Tyr 20 2522 25 PRT TS60 Mutant of Zea mays 22 Glu Asp Val Gly Thr Ile Lys Ser PhePhe Asp Ala Asn Leu Val Leu 1 5 10 15 Thr Glu Gln Pro Ser Lys Phe AspPhe 20 25 23 25 PRT Zea mays 23 Glu Asp Val Gly Thr Ile Lys Ser Phe PheAsp Ala Asn Leu Ala Leu 1 5 10 15 Thr Glu Gln Pro Ser Lys Phe Asp Phe 2025 24 25 PRT Triticum aestivum 24 Glu Asp Ile Gly Thr Ile Arg Ser PhePhe Asp Ala Asn Met Ala Leu 1 5 10 15 Cys Glu Gln Pro Pro Lys Phe GluPhe 20 25 25 25 PRT Hordeum vulgare 25 Glu Asp Ile Gly Thr Ile Arg SerPhe Phe Asp Ala Asn Met Ala Leu 1 5 10 15 Cys Glu Gln Pro Pro Lys PheGlu Phe 20 25 26 25 PRT Oryza sativa 26 Glu Asp Ile Gly Thr Ile Lys SerPhe Phe Asp Ala Asn Leu Ala Leu 1 5 10 15 Thr Glu Gln Pro Pro Lys PheGlu Phe 20 25 27 20 PRT RTS48-2 Mutant of Zea mays 27 Thr Gln Met ProGlu Glu Pro Val Gly Trp Phe Gln Gly Thr Ala Asp 1 5 10 15 Ser Ile ArgLys 20 28 20 PRT Zea mays 28 Thr Gln Met Pro Glu Glu Pro Ala Gly Trp PheGln Gly Thr Ala Asp 1 5 10 15 Ser Ile Arg Lys 20 29 20 PRT Triticumaestivum 29 Thr Gln Met Pro Gly Glu Ala Ala Gly Trp Phe Arg Gly Thr AlaAsp 1 5 10 15 Ala Val Arg Lys 20 30 20 PRT Hordeum vulgare 30 Thr GlnMet Pro Gly Glu Ala Ala Gly Trp Phe Arg Gly Thr Ala Asp 1 5 10 15 AlaVal Arg Lys 20 31 20 PRT Oryza sativa 31 Thr Gln Met Pro Asp Glu Pro AlaGly Trp Phe Gln Gly Thr Ala Asp 1 5 10 15 Ala Ile Arg Lys 20 32 18 PRTRTS60-1 Mutant of Zea mays 32 Asp Lys Cys Lys Met Lys Tyr Val Phe IleSer Asp Gly Cys Leu Leu 1 5 10 15 Arg Glu 33 18 PRT Zea mays 33 Asp LysCys Lys Met Lys Tyr Ala Phe Ile Ser Asp Gly Cys Leu Leu 1 5 10 15 ArgGlu 34 18 PRT Triticum aestivum 34 Asp Lys Cys Arg Ile Lys Glu Ala IleIle Ser His Gly Cys Phe Leu 1 5 10 15 Arg Glu 35 18 PRT Hordeum vulgare35 Asp Lys Cys Arg Ile Lys Glu Ala Ile Ile Ser His Gly Cys Phe Leu 1 510 15 Arg Glu 36 20 PRT Oryza sativa 36 Asp Lys Cys Lys Cys Lys Ile LysAsp Ala Ile Ile Ser Asp Gly Cys 1 5 10 15 Ser Phe Ser Glu 20

We claim:
 1. A purified polynucleotide encoding a mutant large subunitof ADP-glucose pyrophosphorylase polypeptide of maize, or abiologically-active fragment or variant of said mutant polypeptide,wherein when said mutant polypeptide is expressed with the small subunitof ADP-glucose pyrophosphorylase to form a mutant ADP-glucosepyrophosphorylase enzyme, said mutant enzyme, or a biologically-activefragment or variant of said mutant enzyme, exhibits increased heatstability relative to wild type ADP-glucose pyrophosphorylase enzyme. 2.The polynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises an amino acid mutation whereinthe histidine amino acid at position 333 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 3. The polynucleotide according toclaim 2, wherein the amino acid that replaces histidine at positionnumber 333 is a tyrosine.
 4. The polynucleotide according to claim 1,wherein said mutant polypeptide encoded by said polynucleotide comprisesan amino acid mutation wherein the alanine amino acid at position 177 inthe amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 5. Thepolynucleotide according to claim 4, wherein the amino acid thatreplaces alanine at position number 177 is a proline.
 6. Thepolynucleotide according to claim 4, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 7. Thepolynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises an amino acid mutation whereinthe aspartic acid amino acid at position 400 in the amino acid sequenceof the wild type large subunit of ADP-glucose pyrophosphorylasepolypeptide of maize is replaced by an amino acid that confers saidincreased heat stability on said mutant enzyme.
 8. The polynucleotideaccording to claim 7, wherein the amino acid that replaces aspartic acidat position number 400 is a histidine.
 9. The polynucleotide accordingto claim 1, wherein said mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the valine aminoacid at position 454 in the amino acid sequence of the wild type largesubunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 10. The polynucleotide according to claim 9, whereinthe amino acid that replaces valine at position number 454 is anisoleucine.
 11. The polynucleotide according to claim 1, wherein saidmutant polypeptide encoded by said polynucleotide comprises an aminoacid mutation wherein the arginine amino acid at position 104 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 12. Thepolynucleotide according to claim 11, wherein the amino acid thatreplaces arginine at position number 104 is a threonine.
 13. Thepolynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises an amino acid mutation whereinthe threonine amino acid at position 460 in the amino acid sequence ofthe wild type large subunit of ADP-glucose pyrophosphorylase polypeptideof maize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 14. The polynucleotide according toclaim 13, wherein the amino acid that replaces threonine at positionnumber 460 is an isoleucine.
 15. The polynucleotide according to claim1, wherein said mutant polypeptide encoded by said polynucleotidecomprises an amino acid mutation wherein the arginine amino acid atposition 216 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 16. The polynucleotide according to claim 15, wherein the aminoacid that replaces arginine at position number 216 is a proline.
 17. Thepolynucleotide according to claim 1, wherein said mutant polypeptideencoded by said polynucleotide comprises an amino acid mutation whereinthe alanine amino acid at position 396 in the amino acid sequence of thewild type large subunit of ADP-glucose pyrophosphorylase polypeptide ofmaize is replaced by an amino acid that confers said increased heatstability on said mutant enzyme.
 18. The polynucleotide according toclaim 17, wherein the amino acid that replaces alanine at positionnumber 396 is a valine.
 19. The polynucleotide according to claim 1,wherein said mutant polypeptide encoded by said polynucleotide comprisesa first amino acid mutation wherein the leucine amino acid at position426 in the amino acid sequence of the wild type large subunit ofADP-glucose pyrophosphorylase polypeptide of maize is replaced byphenylalanine; and, a second amino acid mutation wherein the alanineamino acid at position 177 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by valine.
 20. The polynucleotide according to claim 1, whereinsaid mutant polypeptide encoded by said polynucleotide comprises a firstamino acid mutation wherein the glutamic acid amino acid at position 324in the amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by lysine; a secondamino acid mutation wherein the alanine amino acid at position 359 inthe amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by valine; and athird amino acid mutation wherein the alanine amino acid at position 396in the amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by valine.
 21. Amethod for increasing heat resistance of a plant, said method comprisingintroducing the polynucleotide of claim 1 into said plant and expressingthe protein encoded by said polynucleotide molecule, thereby resultingin a plant with increased heat resistance.
 22. The method according toclaim 21, wherein said plant is a cereal.
 23. The method according toclaim 22, wherein said cereal is selected from the group consisting ofmaize, wheat, rice, and barley.
 24. The method according to claim 21,wherein said plant is Zea mays.
 25. A transgenic or mutagenized plantcomprising the polynucleotide of claim
 1. 26. The plant according toclaim 25, wherein said plant is a cereal.
 27. The plant according toclaim 26, wherein said cereal is selected from the group consisting ofmaize, wheat, rice, and barley.
 28. The plant according to claim 25,wherein said plant is Zea mays.
 29. The plant according to claim 25,wherein the mutant polypeptide encoded by said polynucleotide comprisesan amino acid mutation wherein the histidine amino acid at position 333in the amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 30. Theplant according to claim 29, wherein the amino acid that replaceshistidine at position number 333 is a tyrosine.
 31. The plant accordingto claim 25, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the alanineamino acid at position 177 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 32. The plant according to claim 31, wherein theamino acid that replaces alanine at position number 177 is a proline.33. The plant according to claim 31, wherein the amino acid thatreplaces alanine at position number 177 is a valine.
 34. The plantaccording to claim 25, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the asparticacid amino acid at position 400 in the amino acid sequence of the wildtype large subunit of ADP-glucose pyrophosphorylase polypeptide of maizeis replaced by an amino acid that confers said increased heat stabilityon said mutant enzyme.
 35. The plant according to claim 34, wherein theamino acid that replaces aspartic acid at position number 400 is ahistidine.
 36. The plant according to claim 25, wherein the mutantpolypeptide encoded by said polynucleotide comprises an amino acidmutation wherein the valine amino acid at position 454 in the amino acidsequence of the wild type large subunit of ADP-glucose pyrophosphorylasepolypeptide of maize is replaced by an amino acid that confers saidincreased heat stability on said mutant enzyme.
 37. The plant accordingto claim 36, wherein the amino acid that replaces valine at positionnumber 454 is an isoleucine.
 38. The plant according to claim 25,wherein the mutant polypeptide encoded by said polynucleotide comprisesan amino acid mutation wherein the arginine amino acid at position 104in the amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 39. Theplant according to claim 38, wherein the amino acid that replacesarginine at position number 104 is a threonine.
 40. The plant accordingto claim 25, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the threonineamino acid at position 460 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 41. The plant according to claim 40, wherein theamino acid that replaces threonine at position number 460 is anisoleucine.
 42. The plant according to claim 25, wherein the mutantpolypeptide encoded by said polynucleotide comprises an amino acidmutation wherein the arginine amino acid at position 216 in the aminoacid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 43. Theplant according to claim 42, wherein the amino acid that replacesarginine at position number 216 is a proline.
 44. The plant according toclaim 25, wherein the mutant polypeptide encoded by said polynucleotidecomprises an amino acid mutation wherein the alanine amino acid atposition 396 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced by anamino acid that confers said increased heat stability on said mutantenzyme.
 45. The plant according to claim 44, wherein the amino acid thatreplaces alanine at position number 396 is a valine.
 46. The plantaccording to claim 25, wherein the mutant polypeptide encoded by saidpolynucleotide comprises a first amino acid mutation wherein the leucineamino acid at position 426 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by phenylalanine; and, a second amino acid mutation wherein thealanine amino acid at position 177 in the amino acid sequence of thewild type large subunit of ADP-glucose pyrophosphorylase polypeptide ofmaize is replaced by valine.
 47. The plant according to claim 25,wherein the mutant polypeptide encoded by said polynucleotide comprisesa first amino acid mutation wherein the glutamic acid amino acid atposition 324 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced bylysine; a second amino acid mutation wherein the alanine amino acid atposition 359 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced byvaline; and a third amino acid mutation wherein the alanine amino acidat position 396 in the amino acid sequence of the wild type largesubunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by valine.
 48. A transgenic or mutagenized plant tissuecomprising the polynucleotide of claim
 1. 49. The plant tissue accordingto claim 48, wherein said plant tissue is from a cereal.
 50. The planttissue according to claim 49, wherein said cereal is selected from thegroup consisting of maize, wheat, rice, and barley.
 51. The plant tissueaccording to claim 48, wherein said plant tissue is from Zea mays. 52.The plant tissue according to claim 48, wherein said plant tissue is aseed.
 53. The plant tissue according to claim 48, wherein the mutantpolypeptide encoded by said polynucleotide comprises an amino acidmutation wherein the histidine amino acid at position 333 in the aminoacid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 54. Theplant tissue according to claim 53, wherein the amino acid that replaceshistidine at position number 333 is a tyrosine.
 55. The plant tissueaccording to claim 48, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the alanineamino acid at position 177 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 56. The plant tissue according to claim 55, whereinthe amino acid that replaces alanine at position number 177 is aproline.
 57. The plant tissue according to claim 55, wherein the aminoacid that replaces alanine at position number 177 is a valine.
 58. Theplant tissue according to claim 48, wherein the mutant polypeptideencoded by said polynucleotide comprises an amino acid mutation whereinthe aspartic acid amino acid at position 400 in the amino acid sequenceof the wild type large subunit of ADP-glucose pyrophosphorylasepolypeptide of maize is replaced by an amino acid that confers saidincreased heat stability on said mutant enzyme.
 59. The plant tissueaccording to claim 58, wherein the amino acid that replaces asparticacid at position number 400 is a histidine.
 60. The plant tissueaccording to claim 48, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the valine aminoacid at position 454 in the amino acid sequence of the wild type largesubunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 61. The plant tissue according to claim 60, whereinthe amino acid that replaces valine at position number 454 is anisoleucine.
 62. The plant tissue according to claim 48, wherein themutant polypeptide encoded by said polynucleotide comprises an aminoacid mutation wherein the arginine amino acid at position 104 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 63. Theplant tissue according to claim 62, wherein the amino acid that replacesarginine at position number 104 is a threonine.
 64. The plant tissueaccording to claim 48, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the threonineamino acid at position 460 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 65. The plant tissue according to claim 64, whereinthe amino acid that replaces threonine at position number 460 is anisoleucine.
 66. The plant tissue according to claim 48, wherein themutant polypeptide encoded by said polynucleotide comprises an aminoacid mutation wherein the arginine amino acid at position 216 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by an amino acid thatconfers said increased heat stability on said mutant enzyme.
 67. Theplant tissue according to claim 66, wherein the amino acid that replacesarginine at position number 216 is a proline.
 68. The plant tissueaccording to claim 48, wherein the mutant polypeptide encoded by saidpolynucleotide comprises an amino acid mutation wherein the alanineamino acid at position 396 in the amino acid sequence of the wild typelarge subunit of ADP-glucose pyrophosphorylase polypeptide of maize isreplaced by an amino acid that confers said increased heat stability onsaid mutant enzyme.
 69. The plant tissue according to claim 68, whereinthe amino acid that replaces alanine at position number 396 is a valine.70. The plant tissue according to claim 48, wherein the mutantpolypeptide encoded by said polynucleotide comprises a first amino acidmutation wherein the leucine amino acid at position 426 in the aminoacid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by phenylalanine;and, a second amino acid mutation wherein the alanine amino acid atposition 177 in the amino acid sequence of the wild type large subunitof ADP-glucose pyrophosphorylase polypeptide of maize is replaced byvaline.
 71. The plant tissue according to claim 48, wherein the mutantpolypeptide encoded by said polynucleotide comprises a first amino acidmutation wherein the glutamic acid amino acid at position 324 in theamino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by lysine; a secondamino acid mutation wherein the alanine amino acid at position 359 inthe amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by valine; and athird amino acid mutation wherein the alanine amino acid at position 396in the amino acid sequence of the wild type large subunit of ADP-glucosepyrophosphorylase polypeptide of maize is replaced by valine.